CN112903874B - Free and conjugated clenbuterol standard substance contained in pig urine and preparation method thereof - Google Patents
Free and conjugated clenbuterol standard substance contained in pig urine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a standard substance containing clenbuterol in free state and conjugated state in pig urine and a preparation method thereof. Feeding animals to obtain a pig urine raw material containing free clenbuterol and conjugated clenbuterol in concentration ranges, and freeze-drying after setting a value to obtain a pig urine freeze-dried powder containing free clenbuterol and conjugated clenbuterol standard substance with uniformity and stability meeting requirements; adding water to re-dissolve and reduce into liquid, pretreating by a mixed biological enzyme enzymolysis method, and determining the clenbuterol content in the matrix standard substance by a liquid chromatography-tandem mass spectrometry/isotope internal standard method combined method. The standard substance provided by the invention reflects the real condition of the clenbuterol standard substance in pig urine after metabolism in an animal body, has more accurate definite value and good uniformity and stability, and can be widely applied to numerous fields such as clenbuterol residue detection in animal urine, quality verification of fast screening products, evaluation of instrument detection methods, quality control in laboratories and laboratory capacity verification ratio.
Description
Technical Field
The invention relates to the technical field of preparation of veterinary drug residue detection standards, and particularly relates to a conjugated clenbuterol standard substance in pig urine freeze-dried powder after animal metabolism and a preparation method thereof.
Background
Clenbuterol is beta 2 One of the receptor agonists, also the first-appearing, most typical representative of the class of substances known colloquially as "clenbuterol", is a toxic, harmful compoundHas the characteristics of low cost and quick effect, is illegally and illegally used in animal breeding after being exposed for many times, and brings serious risks to human health and public health safety. Clenbuterol is always listed as an important parameter for monitoring animal food, and currently, methods for determining clenbuterol residues in edible animal tissues such as pork and pork liver and in pig urine are more, and an instrument method generally adopts a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and a rapid screening method generally adopts an enzyme-linked immunosorbent assay (ELISA) method and a colloidal gold test strip method.
However, as products such as pork and pork liver entering the market are monitored, the hysteresis quality is high, so that the products are difficult to trace and retrieve in time, difficult to treat and destroy problematic products, and great waste is caused. Therefore, aiming at monitoring the quality safety of animal products, the animal urine is a monitoring target substance with the outstanding advantages of convenience, rapidness, directness, low cost, early warning and the like, whether the animal uses prohibited substances or not can be effectively monitored in time on the premise of not slaughtering the animal, thereby more effectively improving the supervision capability of food safety, avoiding the problem products from flowing into the market, eliminating residues through methods such as animal metabolism and the like, reducing unnecessary loss and having extremely strong economic benefit.
At present, no matter fast screening detection such as an ELISA method and a test strip method which are commonly used in a breeding farm and a slaughter house, or confirmation work of detecting the clenbuterol in the pig urine by using an instrument method such as LC-MS/MS and the like in a laboratory, a clenbuterol standard solution is temporarily added into so-called blank pig urine which is preserved for a long time, and a positive sample of the pig urine containing the clenbuterol is prepared in a simulated mode and is used as a quality control sample or a matrix standard sample. However, if the sample obtained by 'temporary preparation' is added by artificial exogenous means and biological enzyme pretreatment is not performed on the urine to be detected, the detection effect of the conventional kit or test strip is adopted on site, which is often inaccurate, and thus false negative results occur. Therefore, the so-called swine urine positive sample which is obtained by 'temporary preparation' and simulates the inclusion of clenbuterol can seriously affect the accuracy, reliability and comparability of a detection result in food safety monitoring work, especially in the field environment where experimental conditions cannot meet strict requirements. The on-site detection result shows that the urine sample which does not exceed the standard possibly exceeds the standard after being pretreated by an enzymolysis method in a laboratory and then being tested by a liquid chromatography-tandem mass spectrometry.
For on-site rapid screening detection, under the condition that pretreatment by enzymolysis cannot be used, in order to meet the requirements of on-site detection, reduce the detection cost and the detection time and improve the accuracy of a detection result, a more accurate and more applicable urine matrix standard product is urgently needed to be provided for correction detection and quality control, so that the accuracy of on-site screening of the clenbuterol content in pig urine can be greatly improved, the accuracy and effective monitoring of clenbuterol in food safety monitoring work can be further enhanced, the verification of market products in a rapid screening detection method is standardized, the evaluation of an instrument method in the detection is confirmed by scientific judgment, and the comparison level of internal quality control and external capability in a laboratory is improved.
Disclosure of Invention
The invention aims to provide a standard substance containing clenbuterol in free state and conjugated state after being metabolized by animals.
In long-term detection and screening, the invention finds that if the same urine sample is calibrated and fixed by adopting a standard substance which is temporarily added and prepared, and the field rapid detection is compared with the detection by adopting a laboratory high performance liquid chromatography-tandem mass spectrometry, the detection result of the sample has larger deviation, which shows that part of the sample shows that the clenbuterol is negative under the field rapid detection condition, and if the sample is detected by the laboratory high performance liquid chromatography-tandem mass spectrometry, the sample shows that the clenbuterol is positive. The inventor tries to detect the positive urine which is subjected to enzyme treatment in advance in a high performance liquid chromatography-tandem mass spectrometry method by using a field rapid kit method in a laboratory, and finds that the results of the two methods tend to be consistent. The step of adding enzyme to pretreat the urine to be detected in the high performance liquid chromatography-tandem mass spectrometry is a key factor for ensuring the consistency of the detection result of the on-site rapid detection and the detection result of the high performance liquid chromatography-tandem mass spectrometry in a laboratory. The invention further researches show that the positive urine contains clenbuterol in a free state and a conjugated state, the existing commonly used standard substance is clenbuterol in pig urine obtained by artificially and externally adding temporary preparation, and the temporarily prepared standard substance is not subjected to the circulating metabolic process in an animal body, so that all clenbuterol in the standard substance is in the free state. In the detection of high performance liquid chromatography-tandem mass spectrometry for laboratories, all clenbuterol in a sample is in a free state due to the use of enzyme pretreatment, so that the detection result is not influenced; however, in the field rapid detection, because no enzyme pretreatment is used, the field rapid detection result is lower than the laboratory detection result, and false negative results are easy to occur.
Therefore, the temporarily prepared standard substance can not be used in the on-site rapid screening test, but a clenbuterol standard substance containing both free state and conjugated state can be used to accurately determine the value in the on-site rapid screening test, otherwise, a false negative sample can flow into the market, and the food safety hazard is caused.
In view of the above, the invention considers that pig urine containing clenbuterol in free state and conjugated state obtained after artificial feeding animal (pig) before slaughter is metabolized is used as a standard substance for the correction standard substance for the on-site rapid detection of clenbuterol. After a large amount of groping and data comparison, the swine urine containing clenbuterol in free state and conjugated state obtained after animal metabolism by the method is found to be used as a standard substance, a fixed value result in on-site rapid detection is consistent with a detection result of a laboratory instrument method, the problem of false negative caused by using a standard substance prepared by temporary addition is avoided, the uniformity and the stability are good, and a combined fixed value of a plurality of laboratories is obtained.
Specifically, the preparation method of the clenbuterol in free state and conjugated state in the pig urine freeze-dried powder after animal metabolism, provided by the invention, comprises the steps of preparing a feed containing clenbuterol to feed live pigs, collecting the urine fed for 72-96h, and uniformly mixing; the concentration of the clenbuterol in the clenbuterol-containing feed is 1-10mg/kg. The inventor proves through long-term experiments that due to differences of individual pigs and different degrees of metabolism, the feed with too low or too high concentration can obtain the pig urine matrix with too low clenbuterol concentration (less than 3 ng/mL) or too high clenbuterol concentration (more than 50 ng/mL). The swine urine with the too low clenbuterol concentration loses the quality evaluation effect on the colloidal gold test strip in the rapid screening detection; the swine urine with too high clenbuterol concentration needs to be diluted by blank urine in the preparation of the later-stage standard substance, and the real proportion of the urine obtained after animal metabolism can be influenced.
Preferably, urine is collected between 72 and 96 hours of feeding.
Preferably, the concentration of clenbuterol in the clenbuterol-containing feed is 5mg/kg. The number of live pigs for preparing the standard substance and collecting urine is not less than 10; the live pigs are live pigs 2-4 weeks before slaughtering.
In one embodiment of the invention, the applicant finds that the following general rule is that clenbuterol can be detected in pig urine after 3 hours of feeding by continuously and uninterruptedly detecting 12 pig urine samples; during 3-72 hours, the concentration of clenbuterol in the pig urine is in a continuous rising period; the content was at a plateau after 72 h. After feeding is stopped, the feed is in a rapid decline period within 3-48 h; after 48h the clenbuterol content in the pig urine was in a slow decline and plateau phase. Through analysis and research on clenbuterol content data obtained by continuously detecting 12 parallel samples, the urine obtained after 72 hours is uniformly mixed after continuously feeding the feed with a certain concentration of clenbuterol, and the content range is 3-30 ng/mL.
Because the existing colloidal gold test strip for rapidly screening and detecting the clenbuterol in the pig urine has the lowest detection limit of 3ng/mL; and tests prove that the clenbuterol concentration in the swine urine after the live swine metabolism within 2-4 weeks after the drug feeding is stopped and before the live swine is slaughtered in the market is detected by an LC-MS/MS method, and the content is 10-20 ng/mL. Therefore, the clenbuterol content concentration in the range is most suitable for direct value determination and preparation of a urine matrix standard substance, is convenient to be used for rapid screening detection of a colloidal gold test strip, and can also be used for LC-MS/MS method detection of laboratory confirmation.
In the preparation method of the conjugated clenbuterol standard substance in the pig urine freeze-dried powder after animal metabolism, after urine is obtained, the uniformly mixed urine is filtered and centrifuged, supernatant liquid is taken and transferred into a brown sample bottle, and the mixture is freeze-dried until the water content is less than 2%. The preparation method of the invention also comprises the steps of carrying out uniformity detection and stability detection on the standard substance, and carrying out definite value and uncertainty evaluation.
The freeze drying process is carried out by a specific auxiliary freeze drying means.
1. Freezing control, the temperature of the first stage is-25 ℃, the set time is 60 minutes, and the duration is 90 minutes
2. The refrigeration set temperature of the water catcher is-50 ℃ and the duration is 20 minutes.
3. The pre-evacuation was 0.2mbar, the alarm vacuum was 0.8mbar, and the duration of the alarm vacuum was 120 minutes.
4. The temperature of the first stage of primary drying was 5 degrees, set for 360 minutes, duration 360 minutes, and vacuum 0.7mbar. The second stage of primary drying was set at 20 degrees, for 60 minutes, duration 180 minutes and vacuum 0.7mbar.
5. The set temperature for the analytical drying was 40 degrees, the set time was 240 minutes, and the duration was 9999 minutes. The total time of lyophilization was approximately 26 hours.
The invention provides a conjugated clenbuterol standard substance in the pig urine freeze-dried powder after animal metabolism, which is prepared by the preparation method. The quantitative value of the standard substance is 12.80-14.43ng, the average value is 13.67ng, wherein the mass ratio of clenbuterol in free state accounts for about 90-95% of the total amount of clenbuterol, and the mass ratio of clenbuterol in conjugated state accounts for about 5-10% of the total amount of clenbuterol. Through uniformity test, the characteristic values of the clenbuterol in the pig urine are tested by F test, and the uniformity of the matrix standard substance is good; according to the long-term stability monitoring result, the pig urine freeze-dried powder standard substance has good stability within 12 months.
The invention provides a detection method suitable for rapidly detecting the content of clenbuterol in pig urine on site, which takes the clenbuterol freeze-dried powder standard substance containing free state and conjugated state as a detection standard substance and does not need to carry out enzymolysis pretreatment on the urine to be detected.
Further, the invention provides application of the conjugated clenbuterol standard substance in the pig urine freeze-dried powder after animal metabolism in pork food safety supervision.
The invention provides application of conjugated clenbuterol standard substance in the prepared pig urine freeze-dried powder after animal metabolism in detecting the clenbuterol content in a pig to be born.
When the free-state clenbuterol standard substance and the conjugated-state clenbuterol standard substance are contained in the swine urine freeze-dried powder after animal metabolism and used, pure water is added for dissolving, vortex oscillation is carried out, standing is carried out for 3-8min after ultrasonic treatment, the swine urine freeze-dried powder is used as a standard substance containing clenbuterol with a fixed value, and when urine to be detected is detected, an enzymolysis pretreatment step on the urine to be detected is not needed.
When the conjugated clenbuterol standard substance in the pig urine freeze-dried powder after animal metabolism is used, a certain volume of water is firstly added for dissolving, vortex, and stand after ultrasonic treatment, so that the clenbuterol standard substance is used as a standard substance containing a fixed value of clenbuterol pig urine.
The invention has the beneficial effects that: compared with the 'simulation standard substance' which is temporarily prepared by adding from an external source and in which clenbuterol is in a free state, the clenbuterol standard substance in the pig urine freeze-dried powder after animal metabolism can be truly reflected, the actual matrix state (the obtained value has a difference of about 5-10% under the conditions of enzymolysis and non-enzymolysis) of clenbuterol in the pig urine is in a clenbuterol state in the animal urine after animal metabolism is better reflected, the uniformity and the stability are good, the blank that no clenbuterol standard substance in the pig urine freeze-dried powder after animal metabolism exists in China is filled, and the clenbuterol standard substance in the animal urine is mainly applied to the fields of clenbuterol residue detection, quality verification of quick screening products, evaluation of an instrument confirmation detection method, quality control in a laboratory, capability comparison in the laboratory and the like, so that the clenbuterol standard substance has excellent social benefit and economic benefit and good application prospect.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
EXAMPLE 1 preparation of candidate Standard substance
In a live pig farm of about 1000 pigs, 12 representative normal fattening period live pigs (4-5 months old, 2-4 weeks before marketing, body weight of about 120 kg) were randomly selected as representative samples of the parallel test. After preparing the feed containing clenbuterol with a certain concentration, the pig is fed regularly. And meanwhile, continuously collecting the pig urine in a clean sample bottle, and determining the content of the clenbuterol. Through continuous and uninterrupted detection of 12 samples of the obtained pig urine sample, the general rule is found that the content of clenbuterol in the pig urine can be detected after the pig urine is fed for 3 hours; during the period of 3-72 hours, the concentration of the clenbuterol in the pig urine is in a continuous rising period; the content was at a plateau after 72 h. After stopping feeding, the feed is in a rapid decline period within 3-48 h; after 48h the clenbuterol content in the pig urine was in a slow decline and plateau phase.
Through analysis and research on clenbuterol content data obtained by continuously detecting 12 parallel samples, the urine obtained after 72 hours is uniformly mixed after continuously feeding the feed with a certain concentration of clenbuterol, and the content range is 3-20 ng/mL. Therefore, the concentration range obtained after the in vivo metabolism of the live pigs is most suitable for directly determining the value and preparing the urine matrix standard substance.
The specific process is as follows: preparing a live pig compound feed containing 1mg/kg-10mg/kg clenbuterol, getting up the live pig every morning, feeding the live pig on an empty stomach, collecting urine fed for 72h, uniformly mixing, filtering, centrifuging, accurately taking 1.0mL of supernate, transferring the supernate into a 5mL brown sample bottle, and freeze-drying until the water content is less than 2%. The freeze drying process is carried out by the following specific auxiliary freeze drying means:
1. and (3) freezing control, wherein the temperature of the first stage is-25 ℃, the time required for adjusting the temperature from room temperature to the set temperature is 60min, and the time for keeping the temperature is 90min.
2. The refrigeration set temperature of the water catcher is-50 ℃ and the duration time is 20min.
3. The pre-vacuum is 0.2mbar, the alarm vacuum is 0.8mbar, and the alarm vacuum duration is 120min.
4. The temperature of the first stage of primary drying was 5 ℃, the time required to adjust from the last temperature to this set temperature was 360min, the duration was 360min, and a vacuum of 0.7mbar was set. The temperature of the second stage of primary drying was set at 20 ℃, the time required to adjust from the last temperature to this set temperature was 60min, the duration was 180min, and a vacuum of 0.7mbar was set.
5. The set temperature for the analysis drying was 40 ℃, the time required for the adjustment from the previous temperature to the set temperature was 240 minutes, and the duration was 80 minutes. The total time for lyophilization was approximately 26 hours.
And after the bottle cap is sealed, immediately transferring to-20 ℃ for cryopreservation, thus obtaining the standard substance containing the clenbuterol swine urine lyophilized powder matrix.
Before use, a certain volume of water is accurately added into a brown sample bottle, vortex is carried out for 20s, ultrasound is carried out for 1min, and standing is carried out, so that the fixed value clenbuterol-containing pig urine can be used.
EXAMPLE 2 homogeneity test
11 packaging units are randomly extracted before, during and after the whole packaging process, randomly extracted samples are numbered from 1 to 11, and 3 subsamples are further extracted in parallel from each randomly extracted unit and are recorded as numbers 1-1, 1-2, 1-3,2-1, 2-2 and 2-3. The uniformity test method adopts the liquid chromatogram-isotope dilution mass spectrometry to determine the result (the specific method parameters are shown in the standard substance fixed value part), the result adopts the variance analysis method to carry out statistical analysis, and the judgment is carried out by comparing the F test value with the F critical value. The results of the uniformity test and the data statistical analysis of the characteristic values of the clenbuterol component in the pig urine are shown in table 1.
TABLE 1 homogeneity test results (ng/mL) for pig urine matrix standards containing clenbuterol
The experimental data show that the characteristic values of the clenbuterol in the pig urine pass through F test, and the matrix standard substance has good uniformity and meets the technical specification requirements. In addition, the sampling volume during the uniformity test of the experiment is 1.0mL, so that 1.0mL is adopted as the minimum sampling volume in the preparation of the matrix standard substance in the item.
Example 3 stability
Long-term stability monitoring studies were conducted at months 0, 1, 3, 6, 9, and 12, respectively. 3 packaging units are randomly taken each time, each unit is parallelly measured three times, and the measuring method is the same as that adopted by uniformity test and is liquid chromatogram-isotope dilution chromatography-mass spectrometry. And taking the average value of the measurement results of the three packaging units as the long-term stability monitoring result, adopting a trend analysis method for result analysis, fitting a straight line by using the monitoring time and the result, and carrying out statistical analysis on the result. The long-term stability monitoring results of the clenbuterol-containing pig urine lyophilized powder standard substance are shown in table 2, and the stability test results are statistically analyzed by fitting a straight line with the detection time and the results and adopting a trend analysis method.
TABLE 2 Long-term stability monitoring results (ng) for clenbuterol-containing lyophilized pig urine powder base standard substance
EXAMPLE 4 valuing
1. Measurement method selection
According to the technical specification requirements of JJF1006-1994 first-level standard substances, the first-level standard substance can be simultaneously set by two methods with different principles or a method of jointly setting values in multiple laboratories. However, the complex matrix standard substance is generally difficult to satisfy simultaneous quantification of two different principle methods, and an absolute measurement method, namely Isotope Dilution Mass Spectrometry (IDMS), and a multi-laboratory joint quantification method, is usually adopted. The project optimizes sample pretreatment and instrument methods by adopting a stable isotope internal standard-based mode, and adopts a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the value.
2. Laboratory instruments and reagents
Liquid chromatography of the agent 1290 (agent, usa); an agent Tripe Auad MS 6495 Mass Spectrometry (Aglient Corp.); METTLER XS105 one ten-thousandth of a balance and METTLER AL104 one ten-thousandth of a day, on average, have been calibrated by the institute of metrology and testing, beijing, inc.; high speed refrigerated centrifuge (Thermo corporation, usa); vortex mixer model Vortex-Genie 2 Vortex-type Vortex mixer (Scientific Industries, USA); BAKERBOND solid phase extractor (Waters, USA); a water bath nitrogen blower (U.S. organization, N-EVAPTM 111, OA-SYSTM water bath heating device).
Methanol, acetonitrile (LC-MS grade, fisher, usa); formic acid (LC-MS grade, sigma, germany); beta-glucuronidase hydrochloride/arylsulfatase (dima, 300U), the laboratory water being deionized water filtered through a Milli-Q water purification system (0.22 μm filter membrane); the clenbuterol purity standard substance is a national second-grade certified standard substance, is numbered as GBW (E) 061890, has the purity of 99.0 percent and the uncertainty of 0.6 percent (k = 2), and is purchased from a national standard substance research center; clenbuterol-D9: the purity was 99.0%, witega laboratories, germany.
3. Preparation of Standard solutions
(1) Clenbuterol standard stock solution
Accurately weighing 10.0mg (accurate to 0.1 mg) of clenbuterol solid standard substance, fully dissolving the clenbuterol solid standard substance by using methanol, fixing the volume in a 10mL brown volumetric flask, preparing a clenbuterol standard stock solution with the concentration of 1mg/mL, storing the standard stock solution at the temperature of minus 20 ℃, and keeping the validity period of 12 months.
(2) clenbuterol-D9 internal standard stock solution
Accurately weighing 1.0mg (accurate to 0.1 mg) of clenbuterol-D9 isotope solid standard substance, fully dissolving with methanol, fixing the volume in a 10mL brown volumetric flask, preparing clenbuterol-D9 standard stock solution with the concentration of 100mg/L, storing at-20 ℃ and keeping the validity period for 12 months.
(3) Clenbuterol standard intermediate liquid
Accurately measuring 1.0mL of clenbuterol standard stock solution, diluting with methanol, quantitatively accommodating in a 100mL brown bottle, preparing clenbuterol standard intermediate solution with the concentration of 10mg/L, storing at-20 ℃, and having the validity period of 6 months.
(4) clenbuterol-D9 internal standard intermediate liquid
Accurately measuring 1.0mL of clenbuterol-D9 internal standard stock solution, diluting with methanol, quantitatively accommodating in a 100mL brown bottle, preparing clenbuterol-D9 standard intermediate solution with the concentration of 1mg/L, storing at-20 ℃ and having the validity period of 6 months.
(5) Standard working fluid
A certain amount of clenbuterol standard intermediate solution is accurately measured respectively, and clenbuterol standard working solution with the concentration of 1, 2, 10, 20 and 50 mu g/L is prepared by diluting the mobile phase respectively.
(6) Isotope labeled working solution
Accurately measuring a certain amount of clenbuterol-D9 standard intermediate solution, diluting with a mobile phase to prepare clenbuterol-D9 standard working solution with the concentration of 200 mu g/L, and preparing the working solution on site.
(7) Mixing calibration solutions
Accurately measuring a certain amount of clenbuterol standard working solution and clenbuterol-D9 standard working solution, mixing, and fixing the volume by using a mobile phase, wherein the concentration is as close as possible to the concentration of a sample on a machine, and the solution is used for single-point calibration and is prepared on site when in use.
4. Sample pretreatment
Extraction: taking a freeze-dried matrix standard sample, adding 1.0mL of water, fully whirling (for a liquid sample, sucking 1.0mL, putting the sample in a 5mL test tube), adding a proper amount of isotope labeled internal standard working solution, adding 0.25mL of 2mol/L ammonium acetate buffer solution (pH 5.2), whirling and oscillating for 1min, adding 40 mu L of beta-glucuronidase/arylsulfatase hydrochloride, and oscillating for 16h in a dark water bath at 37 ℃. Adding 10mL ethyl acetate, mixing for 1min by vortex, centrifuging for 5min at 5000r/min, transferring the supernatant to a 15mL centrifuge tube, blowing nitrogen to dry at 40 ℃, dissolving with 1.0mL 0.1% formic acid water solution, filtering with a 0.22 μm filter membrane, and measuring on a computer.
5. LC-MSMS measuring method
Chromatographic conditions are as follows:
and (3) chromatographic column: c18 column (100 mm. Times.2.1 mm, particle diameter 1.8 μm) or equivalent;
mobile phase a (0.1% aqueous formic acid): mobile phase B (0.1% methanoic acid in methanol) = 1;
flow rate: 0.2mL/min; the sample injection volume is 5 mu L; the column temperature was 30 ℃.
Mass spectrum conditions:
electrospray ion source (ESI); a positive ion scanning mode; multiple Reaction Monitoring (MRM) mode;
ion source temperature: 110 ℃; capillary voltage: 3.5kV;
temperature of drying gas: 350 ℃;
dry gas (nitrogen) flow rate: 450L/h;
collision gas (argon) flow rate: 50L/h.
Other mass spectral parameters are shown in table 3.
TABLE 3 Mass Spectrometry MRM monitoring of clenbuterol ion-pair information
6. Measurement calculation
The LC-MS/MS computer-on sequence is as follows: and finally, calculating the mass concentration of the clenbuterol in the pig urine by adopting a single-point calculation method. The calculation formula is as follows:
C 1 -mass concentration of the test substance in pig urine (ng/mL);
R 1 -the ratio of peak areas of the analyte and the isotopic label in the swine urine sample solution as measured by the instrument;
R 2 -the ratio of the peak areas of the analyte and the isotopic label in the standard working solution measured by the instrument;
M 1 ' -the mass of isotope label added to the pig urine sample (mg);
M 2 ' -mass of isotope label added to standard working solution (mg);
M 2 -mass of standard substance (mg) added to standard working solution;
M S -volume on pig urine sample (mL);
P CRM purity of standard substance.
7. Joint setting value for multiple laboratories
The project develops standard substances containing clenbuterol pig urine freeze-dried powder, adopts a liquid chromatogram-isotope dilution-tandem mass spectrometry (LC-ID-MSMS) as a fixed value measuring method, and combines fixed values in 8 laboratories.
TABLE 4 results of cooperative quantification of standard clenbuterol-containing lyophilized pig urine powder (ng)
In conclusion, the characteristic value of the standard substance containing the clenbuterol lyophilized pig urine powder is determined, and the result is the total average value of 13.67ng in 8 fixed value laboratories, namely the standard value.
Example 5 comparison of results of on-site rapid screening and detection and laboratory instrument detection of clenbuterol standard substance in lyophilized powder of pig urine after animal metabolism and clenbuterol standard substance in temporarily prepared pig urine
The standard substance of clenbuterol pig urine lyophilized powder after animal metabolism (the fixed value of clenbuterol is 3 ng) prepared by the method is added with 1.0mL of water and then redissolved to prepare a calibration solution with the concentration of 3ng/mL, and the calibration solution is used as calibration standard solution 1.
Taking blank pig urine by the prior art, temporarily adding a clenbuterol standard solution to prepare a solution with the concentration of 3ng/mL, and taking the solution as a calibration standard solution 2.
The same swine urine sample (clenbuterol mass concentration of 3ng/mL determined by LC-MS/MS) collected after animal metabolism is subjected to calibration determination by using a calibration standard solution 1 and a calibration standard solution 2 respectively, which is specifically as follows.
1. Measurement method selection
(1) The method comprises the following steps: a laboratory detection method adopts stable isotope as an internal standard, carries out enzymolysis pretreatment on a sample, and carries out value setting by a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
(2) The method 2 comprises the following steps: the on-site rapid screening detection method adopts a common enzyme linked immunosorbent assay kit (ELISA) method according to the on-site rapid screening detection condition, and directly measures the sample without enzymolysis because of on-site detection, and quantifies the sample by using a standard solution enzyme-linked immunosorbent assay curve carried by the kit.
2. Laboratory instruments and reagents
(1) The method comprises the following steps: as in example 4 above.
(2) The method 2 comprises the following steps: enzyme-linked immunosorbent assay (made in China with curve quantitative software); a printer; a micropipette; and a liquid transferring tank.
3. Preparation of Standard solution
(1) The method comprises the following steps: calibration standard solution 1 and calibration standard solution 2.
(2) The method 2 comprises the following steps: calibration standard solution 1 and calibration standard solution 2.
4. Sample pretreatment
(1) The method comprises the following steps: as in example 4 above.
(2) The method 2 comprises the following steps: directly sucking to measure.
5. Measurement method
(1) The method comprises the following steps: as in example 4 above.
(2) The method 2 comprises the following steps: quantitation of microplate reader curves.
6. Calculation of measurement results
(1) The method comprises the following steps: as in example 4 above.
(2) The method 2 comprises the following steps: the microplate reader reads directly.
7. Two methods of constant value comparison
The same samples were subjected to the parallel measurement 7 times by the methods 1 and 2, respectively, and the results are shown in the following tables 5 and 6 after the calibration standard 1 and the calibration standard 2 were evaluated, respectively.
TABLE 5 comparison of results of sample evaluation by method 1 (ng/mL)
| Sample paralleling | Calibration standard solution 1 | Deviation value 1 | Calibration standard solution 2 | Deviation value 2 |
| 1 | 3.01 | +0.01 | 3.06 | +0.06 |
| 2 | 3.04 | +0.04 | 3.04 | +0.04 |
| 3 | 3.02 | +0.02 | 3.05 | +0.05 |
| 4 | 3.05 | +0.05 | 3.05 | +0.05 |
| 5 | 3.02 | +0.02 | 3.02 | +0.02 |
| 6 | 3.03 | +0.03 | 3.06 | +0.06 |
| 7 | 3.04 | +0.04 | 3.07 | +0.07 |
| Mean value of | 3.03 | +0.03 | 3.05 | +0.05 |
TABLE 6 comparison of results of sample evaluation by method 2 (ng/mL)
| Sample paralleling | Calibration standard solution 1 | Deviation value 1 | Calibration standard solution 2 | Deviation value 2 |
| 1 | 3.02 | +0.02 | 2.72 | -0.28 |
| 2 | 3.04 | +0.04 | 2.74 | -0.26 |
| 3 | 3.01 | +0.01 | 2.75 | -0.25 |
| 4 | 3.00 | +0.00 | 2.80 | -0.20 |
| 5 | 3.05 | +0.05 | 2.77 | -0.23 |
| 6 | 3.04 | +0.04 | 2.78 | -0.22 |
| 7 | 3.07 | +0.07 | 2.82 | -0.18 |
| Mean value of | 3.03 | +0.03 | 2.77 | -0.23 |
As is evident by comparing the data in table 5 and table 6:
in the case of method 1 (laboratory instrumental method), since the samples were all pretreated by the enzymatic method, clenbuterol in the conjugated state in urine was also finally free, and therefore, in the case of method 1 (laboratory instrumental method), the final results of the samples measured by the calibration standard solution 1 prepared in this study and the calibration standard solution 2 prepared temporarily were not much different from the actual values of the samples (3 ng/mL) (< 0.05 ng/mL).
However, as for the method 2 (on-site rapid screening method), for the sample, because pretreatment by an enzyme hydrolysis method cannot be performed, part of clenbuterol in a conjugated state in the sample cannot be detected, after the calibration and measurement of the actual sample by using the calibration standard solution 1 prepared in the present study, the result is very close to the results of the method 1 and the actual value (3 ng/mL) of the sample (+ 0.03 ng/mL); the results of calibration measurements on the actual samples using calibration standard solution 2 prepared by the temporary addition were much lower (-0.23 ng/mL) than those obtained by method 1 and the actual values of the samples (3 ng/mL).
Thus, in the case of method 2 (on-site rapid screening method), actual samples obtained after metabolism by the animal would yield results much lower than the actual values if calibrated by the extemporaneous preparation method. Particularly, under the condition that the colloidal gold test strip which is most used in slaughterhouses and farms at present is used for on-site rapid screening detection, the detection limit (3 ng/mL) of clenbuterol in pig urine is judged according to the current colloidal gold test strip, and a sample with the actual content being greater than the detection limit (3 ng/mL) is easy to cause negative (less than 3 ng/mL), so that 'fish missing' under the false negative condition is caused, harmful animal products flow into the market, the health of human bodies is harmed, and potential risk is caused to food safety.
The following conclusions are drawn therefrom:
1. if the standard product (the temporary preparation mode of adding the standard solution to the blank sample) in the prior art is used as the correction solution, the result of the on-site detection is lower than the result of the laboratory instrument detection, and the false negative result of the on-site detection is easily caused.
2. The standard substance after the value setting of the invention is used as the correction fluid, the field detection result and the laboratory result are very close, thereby showing that the standard substance of the invention plays an accurate role and can realize accurate detection without enzyme treatment. The two detection methods in the field and the laboratory have little difference in effect. The standard product of the invention has more practical application value.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A preparation method of clenbuterol standard substance containing free state and conjugated state in pig urine lyophilized powder after animal metabolism is characterized in that feed containing clenbuterol is prepared and fed to live pigs, urine fed for 72-96h is collected and mixed evenly; the concentration of the clenbuterol in the clenbuterol-containing feed is 1-10mg/kg;
the mass ratio of free clenbuterol to free clenbuterol in the clenbuterol-containing and conjugated clenbuterol pig urine freeze-dried powder standard substance after animal metabolism accounts for 90-95% of the total amount of clenbuterol, the mass ratio of conjugated clenbuterol accounts for 5-10% of the total amount of clenbuterol, and the quantitative value of the standard substance is 12.80-14.43ng.
2. The method of claim 1, wherein the number of live pigs is not less than 10, and the live pigs are 2-4 weeks before marketing.
3. The preparation method according to any one of claims 1-2, characterized in that the mixed urine is filtered and centrifuged, the supernatant is taken and transferred into a brown sample bottle, and the mixture is freeze-dried until the water content is less than 2%; and/or carrying out uniformity detection and stability detection on the standard substance of the pig urine freeze-dried powder, and carrying out definite value and uncertainty evaluation.
4. The free and conjugated clenbuterol-containing lyophilized pig urine standard substance obtained by the preparation method of any one of claims 1-3 after animal metabolism.
5. A detection method suitable for rapidly detecting the content of clenbuterol in pig urine on site is characterized in that the standard substance containing free clenbuterol and conjugated clenbuterol lyophilized powder of claim 4 is used as a standard substance for detection of a fixed value, and an enzymolysis pretreatment step for the urine to be detected is not needed.
6. A method for improving the accuracy of on-site rapid detection of clenbuterol content in pig urine is characterized in that the standard substance containing free clenbuterol and conjugated clenbuterol lyophilized pig urine powder of claim 4 is used as a standard substance for detection of a fixed value, an enzymolysis pretreatment step is not required for the urine to be detected, and a detection kit or a test strip is used for detection to detect the clenbuterol content in the pig urine to be detected.
7. The use of the free and conjugated clenbuterol-containing lyophilized pig urine standard substance according to claim 4 for pork food safety supervision or for detecting the clenbuterol content in pigs to be slaughtered.
8. The application of claim 7, wherein when the animal metabolized pig urine lyophilized powder contains free clenbuterol standard substance and conjugated clenbuterol standard substance for use, pure water is added for dissolution, vortex oscillation is performed, standing is performed for 3-8min after ultrasonic treatment, the obtained product is used as a standard substance containing fixed clenbuterol pig urine, and when urine to be detected is detected, an enzymolysis pretreatment step for the urine to be detected is not needed.
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